1. The use of Artemisia annua in the prevention of necrotic enteritis in a broiler disease model R.M. Engberg, K. Grevsen, E. Ivarsen, X. C. Fretté, L.P. Christensen

2. Artemisia annua Common names – Sweet wormwood, Quinghao Pharmacological properties – Antimalarial, antibacterial, anticancer Antimalarial properties Artemisinin and derivatives Artemisinin, Artesunate, Artemether Antibacterial properties Essential oil components Camphor, germacrene D, 1,8-cineole,  - caryophyllene….

3. Hypothesis of the project Necrotic enteritis (NE) Bacterial exotoxins produced by Clostridium perfringens type A (Gram positive anaerobic bacterium) Coccidial infection as a predisposing factor for NE, caused by Eimeria spp., unicellular parasites, belonging to Sporozoa like Plasmodium falciparum (malaria) Artemisia annua Antibacterial activity attributed to essential oil components Antiparasitic activity attributed to Artemisinin and derivates Artemisia annua could possibly be used as feed additive to prevent NE in poultry

4. In vitro antimicrobial activity of Artemisia annua extracts towards Clostridium perfringens 3 extracts tested on Clostridium perfringens strain isolated from diseased poultry flock Extraction of A. annua with hexane Extraction of A. annua with dichloromethane Extraction of A. annua with methanol Solvent extractMIC (ppm) Hexane170 Dichloromethane270 Methanol> 600

5. Experimental design 320 day old broilers (Ross 308) 4 treatments (4 replicate pens à 20 broilers) Group 1: No Artemisia annua supplementation, no infection Group 2: No Artemisia annua supplementation, infection Group 3: With Artemisia annua dried plant material (10 g/kg), infection) Group 4: With Artemisia annua hexan extract (250 mg /kg ), infection Monitoring Body weight 18, 23 and 26 days Lesion scoring on 5 birds/pen on days 22, 24, 27 Clostridium perfringens counts in caecal content (pooled samples of 5 birds per replicate)

6. Disease model Sudden feed shift to a diet providing 30% fish meal at the expense of soya meal on days 17, 18, 19 and 20. A 10 fold overdose of an attenuated live vaccine against coccidiosis (Paracox 5 ®) on day 18. Overnight culture of Clostridium perfringens (strain 48) mixed in the feed (10 7 /g feed) on days 17, 18, 19 and 20. A single dose of an overnight culture (strain 48) orally on day 21 (10 8 /bird).

7. Scoring of small intestinal lesions Thin and friable intestinal walls Score 0No gross lesions 1Thin and friable wall 2Focal necrosis or ulceration (1 to 5 foci) 3Focal necrosis or ulceration (6 to15 foci) 4Focal necrosis or ulceration (16 or more foci) 5Patches of necrosis (2-3 cm long) 6Diffuse necrosis Focal necrosis or ulceration Diffuse necrosis Scoring system for (Keyburn et al., 2006)

8. Small intestinal lesion scores and Clostridium perfringens numbers in caecal content Control Non- infected Control infected Dried plant 10 g/kg infected n-hexane 250 mg/kg infected Significance Lesion score 22 days0 c1 1.75 a 1.30 a 0.60 b *** 24 days0b0b 3.65 a 2.75 a 2.65 a *** 27 days0 c 1.75 a 1.30 a 0.60 b *** C. perfringens (log cfu/g) 22 days3.27 c 8.31 a 7.92 ab 7.10 b *** 24 days5.45 c 8.53 a 8.30 a 7.90 a ** 27 days2.31 c 6.83 a 7.23 a 6.09 b *** 1 Means in the same row with different superscripts differ significantly (P<0.05)

9. Body weight gain through the infection period (day 18 - day 27)

10. Conclusion Using the present disease model, none of the feed additives can prevent small intestinal lesions related to necrotic enteritis. The n-hexane extract of Artemisia annua modulates the course of the disease in terms of a later disease onset and an earlier recovery. The n-hexane extract of Artemisia annua can to a certain extent prevent severe growth depression related to the disease.